DESCRIPTION: The long term specific aim is to elucidate at the atomic level the mechanisms by which binding energy from enzyme-substrate interactions is utilized to facilitate enzymatic catalysis. This objective will be pursued through a global approach utilizing the methods of chemistry, kinetics, spectroscopy, site-directed mutagenesis, and X-ray crystallography. The metabolism of galactose presents fundamental questions bearing on this issue; moreover, galactose metabolism is essential in all living cells, which must break galactose down for use as a fuel and also produce galactose for the biosynthesis of glycoconjugates. One manifestation of the importance of galactose metabolism is the metabolic defect underlying galactosemia, in which galactose metabolism is impaired by structural defects in hexose-1-P uridylyltransferase. In the current grant period, research on UDP- galactose 4-empimerase (epimerase), hexose-1-P uridylyltransferase (uridylyltransferase), and galactose mutarotase (mutarotase) will be emphasized. The enzymes from E. coli will be studied as models for the mammalian enzymes, which are homologous. The specific objectives for epimerase include the elucidation of the structural and kinetic basis for the effect of binding interactions between the UDP-moiety of substrates and the enzyme in enhancing the chemical reactivity of NAD+, the coenzyme that facilitates epimerization at glycosyl C-4 of substrates. Another epimerase objective is to apply structural tests to the prevailing hypothesis accounting for nonstereospecific hydrogen transfer. The glycosyl binding subsite and the catalytic general base/acid within this site will be identified. The structural basis for a charge-transfer interaction between NAD+ and the enzyme will be elucidated. The objectives for research on uridylyltransferase include the structural characterization of the active site in both the resting enzyme and the covalent uridylyl-enzyme will be measured and compared with those for model uridylyl imidazolates to determine the effect of enzymatic binding ont he stability of the intermediate. The glycosyl binding subsite will be identified and characterized. The roles of Zn and Fe ions bound to this enzyme will also be investigated. The current structure reveals the identities of the ligands to these metals to be the side chains of four histidines, two cysteines, and one glutamate. The human enzyme is homologous but lacks one histidine corresponding to His115 that coordinates to Zn in the E. coli enzyme. The human sequence of SerAsp will be introduced into the E. coli enzyme and the structural and kinetic consequences evaluated. Research on the galactose mutarotase will be initiated to determine its chemical mechanism and structure. The enzyme will be purified and subjected to crystallization trials. No mutarotase mechanism has been established and four possible mechanisms will be tested with the objective of eliminating all but one. This one will then be characterized by the application of structural, chemical, kinetic, and spectroscopic techniques.